The long term goal of this project is to understand the functional organization of the vaccinia virus genome. The general approach taken is the isolation and characterization of temperature sensitive and drug resistant mutants of vaccinia virus. This application focuses on the development of procedures designed to generate conditionally lethal mutations in genes which encode enzymes involved in mRNA metabolism, and on detailed characterization of selected mutants isolated previously. Specifically, the aims of this proposal are to l) complete the molecular cloning of the virus DNA and use the clones to map existing mutants by marker rescue, 2) characterize the molecular defect in a mutant, ts22, which displays a specific defect in late virus protein synthesis, 3) complete the characterization of 3 mutations affecting the virus DNA polymerase, 4) characterize the action of 3 specific inhibitors of early vaccinia gene expression (2'-0'methyladenosine, 3'-0'methyladenosine and sinefungin), and study mutants resistant to these drugs and, 5) develop methods for in vitro mutagenesis of specific regions of vaccinia DNA. These studies all generate information useful in understanding virus gene expression, viral and cellular nucleic acid metabolism, and virus host range and pathogenicity. The studies may also be useful for research into the use of vaccinia as a live recombinant DNA vector for vaccination against antigens of foreign origin.